GSM7049172: SUVH1 ChIP in h1 rep2; Arabidopsis thaliana; ChIP-Seq
All the material for ChIP used in this paper were from pooled 10-12 day old seedlings grown on Murashinge and Skoog agar plates (1/2X MS, 1.5% agar, pH5.7). 2-4g of seedling tissue was used for each sample. Samples were ground in liquid nitrogen and crosslinked for 10 or 12 minutes at room temperature in Nuclei Isolation buffer (50mM Hepes pH8, 1M sucrose, 5mM KCl, 5mM MgCl2, 0.6% triton X-100, 0.4 mM PMSF, 5 mM benzamidine hydrochloride, cOmplete EDTA-free Protease Inhibitor Cocktail (Roche)) containing 1% formaldehyde. The crosslinking reactions were stopped with 125 mM glycine by incubation at room temperature for 10 minutes. Crosslinked nuclei were filtered through one layer of miracloth, washed with Extraction Buffer 2, centrifuged through a layer of Extraction Buffer 3 and lysed with Nuclei lysis buffer (buffer compositions are described in the published protocol (Villar and Kohler, 2018)). Chromatin was sheared using a Bioruptor Plus (Diagenode) (20 cycles of 30 seconds on and 30 seconds off) and immunoprecipitated overnight at 4°C with antibody. anti-NRPE1 (endogenous antibody, described in (Liu et al., 2018)), anti-H1 (AS11 1801 Agrisera), anti-H3K9me2 (abcam1220), anti-H3 (abcam1791), anti-FLAG (for both the SUVH1-3xFLAG and the DMS3-ZF ChIPs, we used Anti-FLAG M2 (Sigma, F1804)). 25 µl each of Protein A and Protein G magnetic Dynabeads (Invitrogen) were added to each sample and incubated for 2 more hours at 4°C. The immunoprecipitated chromatin was washed with Low Salt (2X), High Salt, LiCl and TE buffers, for 5 minutes each at 4°